Amplification primers and nucleic acid probes targeted to coccidioides immitis nucleic acid

ABSTRACT

The present invention describes oligonucleotides targeted to Coccidioides immitis nucleic acid sequences which are particularly useful to aid in detecting Coccidioides immitis. The oligonucleotides can aid in detecting Coccidioides immitis in different ways such as by acting as hybridization assay probes, and/or amplification primers.

FIELD OF THE INVENTION

This invention relates to the design and use of oligonucleotidestargeted to Coccidioides immitis nucleic acid. Different types ofoligonucleotides are described including hybridization assay probes,helper probes, and amplification oligonucleotides. The oligonucleotidesare particularly useful for detecting the species Coccidioides immitisin test samples, such as from sputum, tissue samples, body fluids,experimental solutions and cultures.

BACKGROUND OF THE INVENTION

Coccidioides immitis is the etiologic agent of the fungal diseasecoccidioidomycosis (San Joaquin Valley Fever). Infection in man andother animals usually occurs following inhalation of arthroconidia intothe lungs. Disease may be evident after an incubation period of one tofour weeks. Approximately 60 percent of those infections areasymptomatic or characterized by a self-limiting upper respiratoryinfection. The remaining 40 percent of infections proceed to the lowerrespiratory tract resulting in mild or severe pneumonia which mayresolve spontaneously or progress to form pulmonary nodules or cavities,occasionally resembling tuberculosis or carcinoma. In rare cases, theinfection may disseminate to almost any organ of the body, including theskin, bone and central nervous system. Recent increases in infections byfungal pathogens, including Coccidioides immitis have increased the needfor a rapid and sensitive method of detection for Coccidioides immitis.See William A. Check, CAP Today, Aug. 1994, 1, 12-16.

Conventional laboratory identification methods used to identify C.immitis include culture on fungal media, growth rate, colony morphology,microscopic morphology, animal inoculation and biochemical tests. Thesetests may require long incubation periods and require additionalconfirmatory tests. Identification begins with culture of the specimenon fungal media. The time required for growth to a visible, cobweb-likecolony varies from 3 to 21 days and the mature colony morphology varies.Additional growth is needed before the characteristic microscopicsporulation pattern of alternating arthroconidia may be seen. Manyspecies of fungi other than C. immitis may produce similar colony andsporulation patterns, including such naturally occurring soil fungi asMalbranchea and Uncinocarpus spp. Some yeast-like organisms such asGeotrichum and Trichosporon spp. may also resemble C. immitis.

Animal inoculation is another method sometimes used to detectCoccidioides immitis by producing the species-specific spherulescharacteristic of Coccidioides immitis. Still other confirmatory testsbased on exoantigen extraction have been described, but these tests maytake 3 to 5 days or longer to perform.

None of the references herein are admitted to be prior art.

SUMMARY OF THE INVENTION

This invention concerns oligonucleotides targeted to Coccidioidesimmitis nucleic acid sequences, and methods of detecting Coccidioidesimmitis. Hybridization assay probes, amplification primers, and helperprobes are described. Hybridization assay probes can preferentiallyhybridize under stringent hybridization assay conditions to aCoccidioides immitis nucleic acid target region to form a detectableduplex indicating the presence of Coccidioides immitis in a test sample.Amplification primers can be used to prime amplification reactionsproducing Coccidioides immitis target nucleic acid which can be detectedby the probes described herein. Also featured are probe mixes for use inhybridization assays for the detection of Coccidioides immitis.

In a first aspect, the invention features a hybridization assay probehaving one of the following sequences:

SEQ ID NO:21: GCGCCACGGC ATAAGTTCCT TG,

SEQ ID NO:22: CAAGGAACTT ATGCCGTGGC GC,

SEQ ID NO:23: GCGCCACGGC AUAAGUUCCU UG,

SEQ ID NO:24: CAAGGAACUU AUGCCGUGGC GC.

The probe can distinguish Coccidioides immitis from closely relatedphylogenetic neighbors, by preferentially hybridizing to a Coccidioidesimmitis target nucleic acid sequence region under stringenthybridization assay conditions. The hybridization assay probe is usefulfor detecting the presence of Coccidioides immitis and/or fordetermining the quantity of Coccidioides immitis present in a testsample, e.g., samples of sputum, urine, blood, tissue sections, food,soil and water. Other hybridization probes to the 28S rRNA subunit ofCoccidioides immitis have been previously described in Milliman, U.S.Pat. No. 5,284,747 (not admitted to be prior art).

Probes contain a nucleotide sequence perfectly complementary, orsubstantially complementary, to a Coccidioides immitis target sequence.Hybridization assay probes are sufficiently complementary to nucleicacid containing a target sequence to form a detectable hybridprobe:target duplex under stringent hybridization assay conditions. Ahybridization assay probe is preferably between 15 and 100 nucleotidesin length, more preferably between 15 and 50 nucleotides in length. Evenmore preferably the probe is between 15 and 25 nucleotides in length.

Hybridization assay probes are preferably labeled with a reporter groupmoiety such as a radioisotope, a fluorescent moiety, a chemiluminescentmoiety, an enzyme, or a ligand, incorporated into the probe. The moietycan be used to detect or confirm probe hybridization to its targetsequence.

By "preferentially hybridize" is meant that under stringenthybridization assay conditions, hybridization assay probes can hybridizeto their target nucleic acids to form stable probe:target hybridsindicating the presence of the target nucleic acid and do not form asufficient number of stable probe:non-target hybrids to indicate thepresence of a closely related non-target nucleic acid. Thus, the probehybridizes to target nucleic acid to a sufficiently greater extent thanto non-target nucleic acid to enable one skilled in the art toaccurately detect the presence of Coccidioides immitis and distinguishits presence from that of a closely related organism.

Organisms "closely related" to Coccidioides immitis include Malbrancheaalbolutea, Blastomyces dermatitidis, Candida parapsilosis, Histoplasmacapsulatum, Auxarthron thaxteri, Gymnoascus dugwayensis, Aspergillusflavus, Myxotrichum deflexum, Aspergillis niger, Candida krusei, Candidaglabrata, Aspergillis fumigatus, Arachnioites flavoluteus, Oidiodendronechinulatum, Candida albicans, and Malbranchea dendriticus. The mostclinically important, closely related organisms are Blastomycesdermatitidis and Histoplasma capsulatum.

Another aspect of the present invention relates to compositionscontaining a nucleic acid hybrid made up of a hybridization assay probeand a Coccidioides immitis nucleic acid molecule having a nucleic acidsequence substantially complementary thereto. The hybrid is a stablenucleic acid structure comprising a double-stranded, hydrogen-bondedregion, preferably 15 to 100 nucleotides in length. Such hybrids includeRNA:RNA, RNA:DNA, or DNA:DNA duplex molecules. The hybridization probepresent in the nucleic acid hybrid will contain one of the followingsequences: SEQ ID NOs: 21-24.

Substantially complementary means that the nucleic acid sequence is ableto preferentially hybridize under stringent hybridization assayconditions to a target nucleic acid region. Preferably, the probe has aregion of 9 out of 10 bases which are complementary to the correspondingtarget region. More preferably, the probe has a region of 14 out of 17bases which are complementary to the corresponding target region.

Another aspect of the invention features probe mixes containing ahybridization probe and a helper probe for use in a hybridization assay.Helper probes can be used to facilitate hybridization of a hybridizationassay probe to its target sequence region. Helper probes facilitatehybridization by enhancing the kinetics and/or the Tm of thetarget:hybridization probe duplex. Helper probes are generally describedin Hogan and Milliman, U.S. Pat. No. 5,030,557, which is herebyincorporated by reference herein. In a preferred embodiment, the helperoligonucleotides of the probe mixes comprise, consist essentially of,consist of or are substantially similar to the helper probe sequence:

SEQ ID NO:25: GAACAGGACG TCATAGAGGG TGAGAATCC

or its RNA equivalent (SEQ ID NO:27). The hybridization assay probe ofthe probe mix will contain one of the following sequences: SEQ ID NOs:21-24.

"RNA and DNA equivalent nucleotides" refer to RNA and DNA moleculeshaving the equivalent base pair hybridization properties. RNA and DNAequivalents have different sugar groups (i.e., ribose versus deoxyribose), and may differ by the presence of uracil in RNA and thymine inDNA. The difference between RNA and DNA equivalents do not contribute todifferences in substantially similar nucleic acid sequences.

With respect to a hybridization assay probe or a helper probe, a"substantially similar" nucleotide sequence is a nucleotide sequenceidentical to, or having no more than a 10% nucleotide base differencethan an identified nucleotide sequence (excluding substitution of a RNAor DNA equivalent nucleotide, e.g., substituting T for U or U for T) andwhich enables the probe to hybridize to Coccidioides immitis nucleicacid under stringent hybridization conditions used to detectCoccidioides immitis. With respect to amplification oligonucleotides, a"substantially similar" nucleotide sequence is a nucleotide sequenceidentical to, or having no more than a 10% nucleotide base differencethan an identified nucleotide sequence (excluding substitution of a RNAor DNA equivalent nucleotide) and which enables an amplificationoligonucleotide to prime the amplification of Coccidioides immitistarget nucleic acid under amplification conditions. In alternateembodiments, substantially similar for a hybridization assay probe,helper probe, or amplification oligonucleotide refers to a 5% differencein complementarity to an oligonucleotide containing a particularnucleotide sequence.

The phrases "consists essentially of" or "consisting essentially of"mean that the oligonucleotide has a nucleotide sequence substantiallysimilar to a specified nucleotide sequence and is preferably no morethan four additional nucleotides longer or two nucleotides shorter.Thus, these phrases contain both a sequence length limitation and asequence variation limitation. Any additions or deletions arenon-material variations of the specified nucleotide sequence which donot prevent the oligonucleotide from having its claimed property. Forinstance, with respect to hybridization probes and helper probes, anyadditions or deletions would not prevent the hybridization probes orhelper probes from being able to preferentially hybridize understringent hybridization assay conditions to its target nucleic acid overnon-target nucleic acids. With respect to an amplificationoligonucleotide, any additions or deletions would not prevent theamplification oligonucleotide from being able to hybridize toCoccidioides immitis nucleic acid under amplification conditions, or toprime amplification reactions producing target Coccidioides immitisnucleic acid under amplification conditions.

In another aspect, the invention features amplification oligonucleotidesuseful for amplifying Coccidioides immitis target regions. Amplificationoligonucleotides are preferably 15 to 100 nucleotides in length, morepreferably 15 to 60 nucleotides. Amplification oligonucleotides may havemodifications, such as blocked 3' termini.

Amplification oligonucleotides can act as primers and may be part ofpromoter-primer combinations, i.e., a primer having a specific nucleicacid sequence attached to the 5' terminus that is recognized by an RNApolymerase (including, but not limited to, the promoter sequence for T7,T3, or SP6 RNA polymerase, or sequences enhancing initiation orelongation of RNA transcription by an RNA polymerase). One example of apromoter sequence includes the sequence SEQ ID NO: 41 5'-AATTTAATACGACTCACTAT AGGGAGA-3'. Other examples of useful promoter sequences arecontained in various commercially available vectors including, forexample, pBluescript® vectors from Stratagene Cloning Systems or thepGEM™ vectors from Promega Biotec.

Preferably, amplification oligonucleotides contain a primer sequencehaving, consisting essentially of, consisting of, or substantiallysimilar to one of the following sequences:

SEQ ID NO:9: GCTCAAATTT GAAATCTGTC CATGCGGAGC

SEQ ID NO:11 (RNA equivalent to SEQ ID NO:9),

SEQ ID NO:29: GTCCAGCAGC CACAGACGGG ATTC,

SEQ ID NO:31 (RNA equivalent to SEQ ID NO:29),

SEQ ID NO:33: CACAGACGGG ATTCTCACCC TC,

SEQ ID NO:35 (RNA equivalent to SEQ ID NO:33),

SEQ ID NO:37: GGATTCTCAC CCTCTATGAC GTCCTG, and

SEQ ID NO:39 (RNA equivalent to SEQ ID NO:37).

More preferably, the amplification oligonucleotide will contain a primersequence corresponding to SEQ ID NOs: 33, 35, 37, 39. Even morepreferably, the amplification oligonucleotide will contain a primersequence corresponding to SEQ ID NOs: 37, 39.

Examples of amplification oligonucleotides having a promoter sequenceare:

SEQ ID NO:1: AATTTAATAC GACTCACTAT AGGGAGAGTC CAGCAGCCAC AGACGGGATT C,

SEQ ID NO:5: AATTTAATAC GACTCACTAT AGGGAGACAC AGACGGGATT CTCACCCTC, and

SEQ ID NO:13: AATTTAATAC GACTCACTAT AGGGAGAGGA TTCTCACCCT CTATGACGTCCTG.

More preferably, amplification oligonucleotides have, consistessentially of, consist or, or are substantially similar to, sequencesprovided by SEQ ID NOs: 5, 13. Even more preferably, amplificationoligonucleotides have or consist essentially of sequences provided bySEQ ID NO: 13.

Amplification oligonucleotides can be used in nucleic acid amplificationprocedures, such as the polymerase chain reaction or TranscriptionMediated Amplification reaction using RNA polymerase, DNA polymerase andRNaseH or its equivalent, as described by Kacian and Fultz supra, herebyincorporated by reference herein. In addition, other methods of makinguse of transcription in amplification assays are described in Sninsky etal., U.S. Pat. No. 5,079,351.

Amplification oligonucleotides hybridize with a target nucleic acid andmay act as a primer for nucleic acid synthesis. The oligonucleotidesamplified by extension of the primers will be complementary to thehybridization assay probe. Preferably, promoters which are recognized byan RNA polymerase such as T7, T3 or SP6 RNA polymerase are used for thetranscription-based amplification.

Amplification means increasing the number of nucleic acid moleculeshaving at least one target nucleic acid sequence complementary to ahybridization assay probe. In order to increase the amplification ofoligonucleotides containing target sequences, amplification preferablyincludes the production of target-template strands containing adouble-stranded promoter region to serve as templates for RNApolymerase.

In other aspects, methods are described for using the hybridizationassay probes, helper probes and amplification oligonucleotides to detectCoccidioides immitis and to distinguish Coccidioides immitis fromclosely related organisms. These amplification assays preferably involveamplifying target nucleic acid in a sample to be tested, contacting theamplified sequences under stringent hybridization assay conditions witha hybridization assay probe which preferentially hybridizes withCoccidioides immitis nucleic acid over nucleic acids present in closelyrelated organisms, and measuring the amount of hybridized probe.

The sample is preferably food, soil, or water; a clinical sample such assputum, urine, blood, or tissue sections; or nucleic acid isolated froma cultured sample. More preferably, the amplification assay will be usedto detect Coccidioides immitis directly from a clinical sample.Detection directly from a clinical sample means that culture of thesample on fungal media is not carried out prior to detection oramplification.

Preferably the amplification assay utilizes a hybridization probeconsisting of one of the following sequences: SEQ ID NOs:17-24. Helperprobes for use in preferred embodiments of the amplification assay have,or are substantially similar to SEQ ID NO:25 and SEQ ID NO:27.Amplification oligonucleotides which can be used in preferredembodiments of the amplification assay have, consist essentially of,consist of, or are substantially similar to SEQ ID NOs:1, 5, 13. Morepreferably, the amplification oligonucleotides used in the amplificationassay will have, consist essentially of, consist of, or aresubstantially similar to SEQ ID NOs: 5, 13. Even more preferably, theamplification oligonucleotides used in the amplification assay willhave, consist essentially of, consist of, or will be substantiallysimilar to SEQ ID NOs:13. In other embodiments, the amplificationoligonucleotides will preferably have, consist essentially of, consistof, or be substantially similar to SEQ ID NOs:9, 11, 29, 31, 33, 35, 37,39; in addition, these amplification oligonucleotides will preferablycontain a nucleic acid sequence to the 5' terminus which is a promoterfor an RNA polymerase, preferably T7 RNA polymerase. Even morepreferably this added nucleic acid sequence will consist essentially ofSEQ ID NO:41.

The oligonucleotides targeted to Coccidioides immitis offer a rapid,non-subjective method of identification and quantitation of Coccidioidesimmitis by detecting the presence of specific nucleic acid sequencesunique to different species and strains of Coccidioides immitis. Theprobes of this invention can be used in hybridization assays to identifyCoccidioides immitis isolated from culture in less than an hour.Furthermore, use of an amplification step allows detection of C. immitisdirectly from clinical samples in less than three hours.

Combining an amplification step with a hybridization assay in theamplification assay increases the amount of target and can thuseliminate the need for culturing C. immitis and its inherent risk ofinfection to laboratory workers. If a hybridization assay oramplification assay is performed in conjunction with culture tests, theassay can warn laboratory workers of the presence of C. immitis in aculture sample.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Described herein are preferred sequences for hybridization assay probes,helper probes, and amplification oligonucleotides designed to hybridizeto target sequences in Coccidioides immitis rRNA or rDNA. In addition,preferred mixes of hybridization assay probes and helper probes usefulfor detecting Coccidioides immitis are described. Also described arehybrids formed by a hybridization assay probe and a target sequence.Preferred methods for using the probes and amplificationoligonucleotides to detect Coccidioides immitis are included in thisdescription.

I. Construction and Use of Hybridization Assay Probes.

A. Obtaining rRNA Sequences

With the exception of viruses, all prokaryotic organisms contain rRNAgenes encoding RNA homologous to 5S rRNA, 16S rRNA and a larger rRNAmolecule known as 23S rRNA. In the eukaryotes these rRNA molecules arethe 5S rRNA, 18S rRNA and 28S rRNA which are substantially similar tothe prokaryotic molecules. Milliman, U.S. Pat. No. 5,284,747, previouslydescribed nucleic acid probes complementary to particular 28S rRNAsequences obtained from Coccidioides immitis, and is hereby incorporatedby reference herein.

Sequence information was obtained experimentally and from publishedinformation. (See Weisburg, et al., J. Bacteriol 171:6455 (1989).)Experimental information was obtained by isolating and sequencing rRNAfrom various organisms using standard techniques known in the art. Morespecifically, rRNA sequence information was obtained by first usingoligonucleotide primers complementary to conserved regions which varylittle between prokaryotic organisms. The oligonucleotide primers werehybridized to the conserved regions in purified rRNA which were specificto the 28S subunit, and extended with the enzyme reverse transcriptaseand deoxyribonucleotides to produce cDNA. E. g., Lane et al., Proc.Nat'l Acad. Sci. USA 82:6955 (1985).

B. Probe Design

Strands of deoxyribo- ("DNA") or ribo- ("RNA") nucleic acid are formedfrom nucleotide units joined in a specific arrangement, or sequence. Anucleotide subunit contains a "base" structure and is distinguished fromanother nucleotide by the base. Bases include adenine (A), cytosine (C),thymine (T), guanine (G), uracil (U), or inosine (I)).

The structures of the bases in the nucleotides permit certain pairs ofbases to interact with one another through the formation of hydrogenbonds. Generally, A is hydrogen bonded to T or U, while G is hydrogenbonded to C. At any point along the chain, therefore, one may find theclassical base pairs AT or AU, TA or UA, GC, or CG. One may also findAG, GU and other "wobble" or mismatched base pairs. Bases which canhydrogen bond are said to be complementary to one another.

Two single strands of DNA or RNA may specifically align and associate("hybridize") to form a double stranded-structure in which the twostrands are held together by the hydrogen bonds which form between pairsof complementary bases. When a first single-strand of nucleic acidcontains sufficient contiguous complementary bases to a second, andthose two strands are brought together under conditions promoting theirhybridization, double-stranded nucleic acid results. Under appropriateconditions, double-stranded DNA/DNA, RNA/DNA, or RNA/RNA hybrids may beformed. Conditions which decrease the likelihood of forming a givendouble-stranded hybrid are said to be more stringent conditions thanconditions in which hybrid formation is less likely.

A probe is generally a single-stranded nucleic acid sequence which iscomplementary to some degree to a nucleic acid oligonucleotide "targetregion" consisting of a "target sequence" sought to be detected. Theprobe may contain a detectable moiety such as a radioisotope, antigen orchemiluminescent moiety. A background description of the use of nucleicacid hybridization as a procedure for the detection of particularnucleic acid sequences is described by Hogan et al., InternationalPatent Application No. PCT/US87/03009, entitled "Nucleic Acid Probes forDetection and/Or Quantitation of Non-Viral Organisms," herebyincorporated by reference herein.

Using methods known to those skilled in the art, and described herein,variable regions of rRNA sequences from the 28S rRNA of Coccidioidesimmitis were identified. The rRNA molecule exhibits a close relationshipof secondary structure to function. This close relationship is caused inpart by intramolecular hydrogen bonding interactions between differentregions of the rRNA molecule. The hydrogen bond interactions leads tothe formation of a double helix structure whose "strands" are differentregions of the same rRNA molecule. The formation of this double-strandedhelix imposes restrictions on evolutionary changes in the primarysequence so that the secondary structure is maintained. This allows twovery different sequences to be aligned based on the conserved primarysequence and also on the conserved secondary structure elements.Potential target sequences for the hybridization assay probes describedherein were identified by noting variations in the homology of thealigned sequences.

The sequence evolution at each of the variable regions is mostlydivergent. Because of this divergence, corresponding rRNA variableregions of more distant phylogenetic relatives of Coccidioides immitisshow greater differences from Coccidioides immitis rRNA than do therRNAs of phylogenetically closer relatives. Sufficient variation betweenCoccidioides immitis and its clinically important, closely relatedphylogenetic relatives, such as Blastomyces dermatitidis and Histoplasmacapsulatum, was observed to identify preferred target sites and designhybridization assay probes useful for distinguishing between nucleicacids of these organisms. These variable regions were subsequently usedas target sequence regions for hybridization assay probes.

B. Oligonucleotide Probes

The present application describes hybridization use in a specific assayto detect Coccidioides immitis, distinguishing it from closely relatedtaxonomic or phylogenetic neighbors. Also described are amplificationoligonucleotides which can be used to amplify target sequences andhelper probes to facilitate probe:target hybrid formation.

A hybridization assay probe is an oligonucleotide which can distinguishCoccidioides immitis from closely related phylogenetic neighbors, bypreferentially hybridizing to a Coccidioides immitis target nucleic acidsequence region under stringent hybridization assay conditions. Thefeatured probes consist of one of the following sequences: SEQ IDNOs:21-24.

By "oligonucleotide," "nucleotide polymer" or "nucleic acid" is meanttwo or more nucleotide subunits covalently joined together. The sugargroups of the nucleotide subunits may be ribose, deoxyribose, ormodified derivatives thereof such as O-methyl ribose. The nucleotidesubunits may be joined by linkages such as phosphodiester linkages,modified linkages or by nonnucleotide moieties, that do not preventpreferential hybridization of the oligonucleotide to its complementarytarget nucleic acid. Modified linkages include those linkages in which astandard phosphodiester linkage is replaced with a different linkage,such as a phosphothionate linkage, or methylphosphonate linkage.

The probes are isolated nucleic acids. By "isolated nucleic acid" ismeant an oligonucleotide or nucleic acid molecule which is present in aform not found in nature without human intervention (e.g., recombinedwith foreign nucleic acid, isolated, or purified to some extent).Preferably, the isolated nucleic acid probe is present in a preparationwhere it constitutes at least 90% of the total nucleic acid presentprior to its use.

The probes may also contain additional nucleotides complementary, or notcomplementary, to nucleic acid sequences contiguous to the targetregion, so long as such additional nucleotides do not preventhybridization to the target region, and in the case of hybridizationassay probes do not prevent preferential hybridization.Non-complementary sequences, such as a promoter sequence, a binding sitefor RNA transcription, a restriction endonuclease recognition site, orsequences which will confer a desired secondary or tertiary structuresuch as a catalytic active site can be used to facilitate detectionand/or amplification.

As illustrated by examples described below, the described hybridizationassay probes can detect Coccidioides immitis and distinguish it fromMalbranchea dendriticus and Oidiodendron echinalatum. The probes canalso distinguish Coccidioides immitis from other closely relatedtaxonomic or phylogenetic neighbors, such as Blastomyces dermatitidisand Histoplasma capsulatum.

C. Hybridization

Hybridization assay probes and helper probes hybridize to their targetsequence under stringent hybridization conditions. Oligonucleotidesacting as helper probes or amplification oligonucleotides do not need tobe able to preferentially hybridize to Coccidioides immitis nucleicacid.

Preferential hybridization of hybridization assay probes to their targetnucleic acids can be accomplished by choosing the appropriatehybridization assay conditions and proper probe design. The stability ofthe probe: target nucleic acid hybrid should be chosen to be compatiblewith the assay and washing conditions so that stable, detectable hybridsform only between nucleic acids having highly complementary sequences.Manipulation of one or more of the different assay parameters determinesthe exact sensitivity and specificity of a particular hybridizationassay probe.

Preferential hybridization can occur under stringent hybridization assayconditions. In general, reducing the degree of complementarity of anoligonucleotide targeted region to its target sequence region decreasesthe degree or rate of hybridization of the probe oligonucleotide to itstarget sequence region. However, additional non-complementarynucleotide(s) may facilitate the ability of an oligonucleotide todiscriminate against non-target organisms.

Preferential hybridization can be measured using techniques known in theart and described herein, such as in the examples provided below.Preferably, there is at least a 100-fold difference between target andnon-target hybridization signals, more preferably at least a 1000-folddifference, even more preferably at least a 10,000-fold difference. Alsopreferably, non-target hybridization signals are not more thanbackground level.

The following guidelines are useful for designing probes and determiningspecific stringent hybridization assay conditions. Because thesensitivity and specificity of hybridization reactions such as thosedescribed herein are affected by a number of factors, including thehybridization assay probe nucleotide sequence and length, the sequenceof the target sequence region, the degree of homology between the targetsequence and the analogous ribosomal nucleic acid sequences from closelyrelated organisms, the hybridization temperature, and the composition ofhybridization reagents, the manipulation of one or more of those factorswill determine the exact sensitivity and specificity of a particularprobe, whether perfectly complementary to its target or not. Theimportance and effect of various hybridization assay conditions,explained further herein, are known to those skilled in the art.

First, the stability of the probe:target nucleic acid hybrid should bechosen to be compatible with the assay and washing conditions so thatstable, detectable hybrids form only between nucleic acids having highlycomplementary sequences. Probes should be designed to have anappropriate melting temperature (Tm). This may be accomplished byvarying the probe length and nucleotide composition (percentage of G+Cversus A+T). The probe length and nucleotide composition are preferablychosen to correspond to a Tm about 2°-10° C. higher than the temperatureat which the final assay will be performed. For instance, the Tm can beincreased by avoiding long A and T rich sequences, or by terminating thehybrids with G:C base pairs. The beginning and end points of the probeshould be chosen so that the length and % G and % C result in a Tm about2°-10° C. higher than the temperature at which the final assay will beperformed.

In general, the optimal hybridization temperature for an oligonucleotideis approximately 5° C. below the melting temperature for a given duplex.Incubation at temperatures below the optimum temperature may allowmismatched base sequences to hybridize and can therefore decreasespecificity. The longer the oligonucleotide, the more base pairs arepresent to hydrogen bond and, in general, the higher the Tm. The basecomposition of the probe is significant because G-C base pairs exhibitgreater additional hydrogen bonding and therefore greater thermalstability than A-T base pairs. (See, e.g., 2 Sambrook, et al., MolecularCloning: A Laboratory Manual 11 (2d ed. 1989) [hereinafter MolecularCloning].) Thus, hybridization involving complementary nucleic acids ofhigher G-C content will be stable at higher temperatures.

To ensure specificity of a hybridization assay probe for its target, itis preferable to design probes which hybridize only with target nucleicacid under conditions of high stringency. Under high stringencyconditions only highly complementary nucleic acid hybrids will form.Accordingly, the stringency of the assay conditions determines theamount of complementarity which should exist between two nucleic acidstrands in order to form a hybrid under those conditions. Stringencyshould be chosen to maximize the difference in stability between theprobe:target hybrid and potential probe:non-target hybrids.

In addition, proper specificity may be achieved by minimizing the lengthof the hybridization assay probe having perfect complementarity tosequences of non-target organisms by minimizing the length of perfectcomplementarity to non-target organisms, avoiding G and C rich regionsof homology to non-target sequences, and by constructing the probe tocontain as many destabilizing mismatches to non-target sequences aspossible. Whether a probe sequence is appropriate for detecting only aspecific type of organism depends largely on the thermal stabilitydifference between probe: target hybrids and probe:non-target hybrids.In designing probes, the differences in these Tm values should be aslarge as possible (e.g., at least 2° C. and preferably 5° C. or more).

The length of the target nucleic acid sequence and, accordingly, thelength of the probe sequence can also be important. In some cases, theremay be several sequences from a particular region, varying in locationand length, which will yield probes with the desired hybridizationcharacteristics. In other cases, one sequence may be significantlybetter than another which differs from it merely by a single base. Whileit is possible for nucleic acids that are not perfectly complementary tohybridize, the longest stretch of perfectly homologous base sequencegenerally determines hybrid stability.

Third, regions of the rRNA which are known to form strong internalstructures inhibitory to hybridization are less preferred targetregions. Likewise, probes with extensive self-complementarity should beavoided. If a strand is wholly or partially involved in anintramolecular or intermolecular hybrid it will be less able toparticipate in the formation of a new intermolecular probe:targethybrid. Ribosomal RNA molecules are known to form very stableintramolecular helices and secondary structures by hydrogen bonding. Bydesigning a probe to a region of the target nucleic acid which remainssubstantially single-stranded under hybridization conditions, the rateand extent of hybridization between probe and target may be increased.

Coccidioides immitis target sequences may initially be present as partof a nucleic acid duplex. For example, a genomic rDNA target occursnaturally in a double-stranded form. The polymerase chain reaction (PCR)also gives rise to a double-stranded product. These double-strandedtargets require denaturation prior to hybridization. Appropriatedenaturation and hybridization conditions are known in the art (e.g., E.M. Southern, J. Mol. Biol. 98:503 (1975)).

The rate of hybridization may be measured by determining the C₀ T_(1/2).The rate at which a probe hybridizes to its target is a measure of thethermal stability of the target secondary structure in the probe region.The standard measurement of hybridization rate is the C₀ T_(1/2) whichis measured as moles of nucleotide per liter times seconds. Thus, it isthe concentration of probe times the half-life of hybridization at thatconcentration. This value is determined by hybridizing various amountsof probe to a constant amount of hybrid for a fixed time.

For example, 0.05 pmol of target is incubated with 0.0012, 0.025, 0.05,0.1 and 0.2 pmol of probe for 30 minutes. The amount of hybrid after 30minutes is measured by the Hybridization Protection Assay as describedbelow. The signal is then plotted as a log of the percent of maximumRelative Light Units (RLU) (from the highest probe concentration) versusprobe concentration (moles of nucleotide per liter). RLU are ameasurement of the quantity of photons emitted by the labeled-probemeasured by the luminometer. The C₀ T_(1/2) is found graphically fromthe concentration corresponding to 50% of maximum hybridizationmultiplied by the hybridization time in seconds. These values range from9.0×10⁻⁶ to 9×10⁻⁵ with the preferred values being less than 3.5×10⁻⁵.

Other methods of nucleic acid reassociation can be used. For example,Kohne and Kacian, EP 229442, entitled "Accelerated Nucleic AcidReassociation Method," describes a method to accelerate nucleic acidreassociation.

A preferred method to determine Tm measures hybridization using ahybridization protection assay (HPA) according to Arnold, et al., U.S.Pat. No. 5,283,171, entitled "Homogeneous Protection Assay." Tm can bemeasured using HPA in the following manner. Probe molecules are labeledwith an acridinium ester. Probe:target hybrids are formed in a lithiumsuccinate buffer (0.1M lithium succinate buffer, pH 5.0, 2 mM EDTA, 2 mMEGTA, 10% (w/v) lithium lauryl sulfate) using an excess amount oftarget. Aliquots of the solution containing the nucleic acid hybrids arethen diluted in the lithium succinate buffered solution and incubatedfor five minutes at various temperatures starting below that of theanticipated Tm (typically 55° C.) and increasing in 2°-5° increments.This solution is then diluted with a mild alkaline borate buffer (0.15Msodium tetraborate, pH 7.6, 5% (v/v) TRITON® X-100) and incubated at alower temperature (for example 50° C.) for ten minutes.

Under these conditions the acridinium ester attached to thesingle-stranded probe is hydrolyzed, while the acridinium ester attachedto hybridized probe is relatively protected from hydrolysis. Thus, theamount of acridinium ester remaining after hydrolysis treatment isproportional to the number of hybrid molecules. The remaining acridiniumester can be measured by monitoring the chemiluminescence produced fromthe remaining acridinium ester by adding hydrogen peroxide and alkali tothe solution. Chemiluminescence can be measured in a luminometer (e.g.,the Gen-Probe LEADER® I or LEADER ®50). The resulting data is plotted aspercent of maximum signal (usually from the lowest temperature) versustemperature. The Tm is defined as the temperature at which 50% of themaximum signal remains. In addition to the method above, Tm may bedetermined by isotopic methods known to those skilled in the art (seee.g., Hogan et al., supra).

The Tm for a given hybrid varies depending on the nature of thehybridization solution used. Factors such as the concentration of salts,detergents, and other solutes can affect hybrid stability during thermaldenaturation (see J. Sambrook, et al., supra). Conditions such as ionicstrength and incubation temperature under which a probe will be usedshould be taken into account in constructing a probe. It is known thatthe thermal stability of a hybrid nucleic acid increases with the ionicstrength of the reaction mixture. On the other hand, the addition ofchemical reagents which disrupt hydrogen bonds, such as formamide, urea,DMSO and alcohols, can greatly reduce hybrid thermal stability andthereby increase the stringency of hybridization. In general, optimalhybridization for synthetic oligonucleotide probes of about 10-50 basesin length occurs approximately 5° C. below the melting temperature for agiven duplex. Incubation at temperatures below the optimum may allowmismatched base sequences to hybridize and can therefore result inreduced specificity.

Examples of specific stringent hybridization conditions forhybridization assay probes are provided in the examples described below.Additional sets of stringent hybridization conditions can be determinedbased on the present disclosure by those of ordinary skill in the art.

D. Oligonucleotide Synthesis

Defined oligonucleotides may be produced by any of several well knownmethods, including automated solid-phase chemical synthesis usingcyanoethylphosphoramidite precursors. Barone et al., Nucleic AcidsResearch 12:4051 (1984). In addition, other well-known methods forconstruction of synthetic oligonucleotides may be employed. MolecularCloning, supra (2:11). Following synthesis and purification of anoligonucleotide, several different procedures may be utilized todetermine the acceptability of the oligonucleotide in terms of size andpurity. Such procedures include polyacrylamide gel electrophoresis andhigh pressure liquid chromatography, both of which are known to thoseskilled in the art.

Once synthesized, selected oligonucleotide hybridization assay probesmay also be labeled with a reporter group by an of several well knownmethods. Molecular Cloning, supra (2:11). Useful labels includeradioisotopes as well as non-radioactive reporting groups. Isotopiclabels include ³ H, ³⁵ S, ³² P, ¹²⁵ I, Cobalt and ¹⁴ C. Isotopic labelscan be introduced into an oligonucleotide by techniques known in the artsuch as nick translation, end labeling, second strand synthesis, reversetranscription, and by chemical methods. When using radio-labeled probes,hybridization can be detected by autoradiography, scintillationcounting, or gamma counting. The chosen detection method depends on thehybridization conditions and the particular radio-isotope used forlabeling.

Non-isotopic materials can also be used for labeling, and may beintroduced internally between nucleotides or at an end of theoligonucleotide. Modified nucleotides may be incorporated enzymaticallyor chemically. Chemical modifications of the probe may be performedduring or after synthesis of the probe, for example, by the use ofnon-nucleotide linker groups as described by Arnold et al., entitled"Non-Nucleotide Linking Reagents for Nucleotide Probes," EPO applicationnumber 88308766.0, publication number 313219 [hereinafter Non-NucleotideLinking Reagents], hereby incorporated by reference herein. Non-isotopiclabels include fluorescent molecules, chemiluminescent molecules,enzymes, cofactors, enzyme substrates, haptens or other ligands.

Preferably, the hybridization assay probes are labeled with anacridinium ester. Acridinium ester labeling may be performed asdescribed by Arnold et al., U.S. Pat. No. 5,185,439 entitled "AcridiniumEster Labeling and Purification of Nucleotide Probes" issued Feb. 9,1993 and hereby incorporated by reference herein.

II. Hybrids Containing a Hybridization Assay Probe and a CoccidioidesImmitis Target Sequence.

Another aspect of this invention is a hybrid formed by a hybridizationassay probe and a target sequence from Coccidioides immitis. The formedhybrid is useful for detecting the presence of target. For example,acridinium ester ("AE") present in hybrid is resistant to hydrolysis inalkali solution while acridinium ester present in single-strandednucleic acid is hydrolyzed in alkali solution. Thus, binding ofAE-labeled probe to target can be detected, after hydrolysis of theunbound AE-labeled probe, by measuring chemiluminescence of acridiniumester remaining in the nucleic acid hybrid. Additionally, the formedhybrid can be used to purify hybridized target from unhybridized probe,thereby removing background due to unhybridized probe. For example,hybrid molecules can be selectively retained on hydroxyapatite columnsor filters using methods well known to those skilled in the art.

III. Mixes of Hybridization Assay Probes and Helper Probes

Mixes of hybridization assay probes and helper probes can be used in thedetection of Coccidioides immitis. Helper probes are used to enhance therate of nucleic acid hybridization of an assay probe with its targetnucleic acid and to facilitate the hybridization of the hybridizationassay probe to its target. In addition, helper probes are sufficientlycomplementary to their target nucleic acid sequence to form a helperprobe:target duplex under stringent hybridization assay conditions. Thestringent hybridization assay conditions used with a given helper probeare determined by the conditions in which a hybridization assay probe isused to preferentially hybridize to its target sequence.

Regions of single-stranded RNA and DNA can be involved in secondary andtertiary structures even under stringent hybridization assay conditions.Such structures can sterically inhibit, or even block hybridization of ahybridization assay probe to its target region. Hybridization of thehelper probe alters the secondary and tertiary structure of the targetnucleic acid, thereby rendering the hybridization assay probe targetregion more accessible. As a result helper probes enhance the kineticsand/or the Tm of the target:hybridization probe duplex. Helper probesare generally selected to hybridize to nucleic acid sequences locatednear the hybridization assay probe target region.

Helper probes which can be used with the hybridization assay probes ofthe present invention are targeted to target nucleic acid sequenceregions of the sequence provided by SEQ ID No: 26. The targeted regionin the helper probe which is complementary to the target regionpreferably contains at least 10 nucleotides of which at least 9 out ofthe 10 nucleotides are perfectly complementary to a nucleic acidsequence present in the target region.

V. Amplification Oligonucleotides and Amplification Assay Conditions

Methods of amplifying the number of target sequences in a sample can becombined with the use of probe sequences to increase the sensitivity andapplicability of the detection assay. (Miller, et al., Evaluation ofGen-Probe Amplified Mycobacterium Tuberculosis Direct Test and PCR forDirect Detection of Mycobacterium tuberculosis in Clinical Specimens, J.Clin. Micro. 1994: 393-397; Reddy, et al., Specific amplification ofAspergillus fumigatus DNA by polymerase chain reaction, Mol. Cell.Probes 7: 121-126 (1993)).

Amplification oligonucleotides can act as primers for primer extensionreactions and may also be part of promoter-primer combinations toamplify a Coccidioides immitis target sequence by TMA. Preferably theamplification oligonucleotide will contain a primer sequence having,consisting essentially of, consisting of, or substantially similar to asequence provided by SEQ ID NOs: 29, 31, 33, 35, 37, 39. Morepreferably, the amplification oligonucleotide will contain a primersequence having, consisting essentially of, consisting of, orsubstantially similar to a sequence provided by SEQ ID NO: 33, 35, 37,39. Even more preferably, the amplification oligonucleotide will containa primer sequence having, consisting essentially of, consisting of, orsubstantially similar to a sequence provided by SEQ ID NO: 37, 39.Preferably the amplification oligonucleotide containing a primersequence will have a promoter sequence attached to the 5' terminus ofthe primer sequence. Preferably the promoter sequence will have, consistessentially of, consist of, or be substantially similar to SEQ ID NO:41.

Preferably the amplification oligonucleotides for use in TMA have,consist essentially of, consist of, or are substantially similar to asequence corresponding to SEQ ID NOs: SEQ ID NOs:1, 5, 13. Morepreferably, the amplification oligonucleotide will have, consistessentially of, consist of, or be substantially similar to a sequencecorresponding to SEQ ID NOs: 5, 13. In an even more preferredembodiment, the amplification oligonucleotide will have, consistessentially of, consist of, or be substantially similar to a sequencecorresponding to SEQ ID NO: 13.

The degree of amplification observed with a set of primers orpromoter-primers depends on several factors, including the ability ofthe oligonucleotides to hybridize to their specific target sequences andtheir ability to be extended or recognized by an RNA polymerase. Whileoligonucleotides of different lengths and base composition may be used,more preferred amplification oligonucleotides have target bindingregions of 30-60 bases and a predicted hybrid Tm of about 65° C.

A target nucleic acid sequence present on a nucleic acid molecule can beamplified using a primer amplification oligonucleotide which hybridizesto the anti-sense target strand 3' of the target sequence and apromoter-primer amplification oligonucleotide which hybridizes to thesense strand 3' of the target sequence. Extension of the promoter-primergives rise to RNA copies of the target sequence. Extension of the primeron the anti-sense strand gives rise to copies of the sense strand"template" for the RNA polymerase which recognizes the promoter-primer.Preferably primers have, consist essentially of, consist of, or aresubstantially similar to the sequences provided by SEQ ID NOs: 9, 11.These primers may also contain an additional promoter sequence added tothe 5' terminus of the primer sequence.

The preferred target sites for amplification oligonucleotides areregions greater than about 15 bases in length. The amplified region,defined by the amplification oligonucleotides, is preferably about 350bases, and more preferably within 150 bases.

Parameters affecting probe hybridization such as Tm, complementarity andsecondary structure also affect primer hybridization and thereforeperformance of the amplification oligonucleotides. These considerations,which were discussed above in the section concerning probe design, canbe modified depending upon the amplification conditions. For example,amplification can be carried out under conditions of lower stringencythan diagnostic hybridization assay conditions.

The degree of non-specific extension (primer-dimer or non-targetcopying) can affect amplification efficiency. Primers are preferablyselected to have low self-or cross complementarity, particularly at the3' ends of the sequence. Long homopolymer tracts and high GC content arepreferably avoided to reduce spurious primer extension. Computerprograms are commercially available to aid in this aspect of the design.

Probe mixes containing hybridization assay probes, helper probes, andamplification oligonucleotides can also be used in amplification assaysfor the detection of Coccidioides immitis.

IV. EXAMPLES

Examples are provided below illustrating different aspects andembodiments of the present invention. The examples illustratemethodologies by which oligonucleotides described herein can be obtainedand tested.

Example I

Procedures

Example A

Lysis of Fungal Strains and Clinical Specimens.

Reference fungal strains (Table 1) used in the nucleic acidamplification experiments were obtained from the American Type CultureCollection (ATCC, Rockville, Md.) and cultured on Sabourd's dextroseagar. Clinical specimens were cultured and the fungi were identifiedusing standard culture and identification methods. In addition, fungalcolonies were identified as Coccidioides immitis using the AccuProbe®Coccidioides immitis Culture Identification Test.

Sputum specimens were digested by treating with an equal volume ofN-acetyl-L-cysteine-NaOH (1%) for 15 minutes. 1.5 mL aliquots wereconcentrated by centrifugation at 10,000×g for 5 min. Kent, P. T.,Kubica G. Public Health Mycobacteriology. A Guide for the Level IIILaboratory. Atlanta, Ga.; Centers for Disease Control (1985).Supernatant fluid was decanted, and 50 μl of sediment was added to a TBlysing tube containing 0.17 g of 0.2-0.3 mm glass beads, 110 μl 5% w/vsorbitol, and 300 μl of a solution of 10 mM N-acetyl-L-cysteine-NaOH, 40mM Trizma base, 2 mM disodium EDTA adjusted to pH 8.0 with HCl (TB SDBsolution).

100 μl of fluid of upper chest fluid, lower chest fluid, expressed swabsor abscess fluid was added to 1 ml of flashtrack wash solution (18 mMNaCl, 5.7 mM sodium azide, 5 mM HEPES (0.5 sodium salt), 0.1% w/vSaponin, adjusted to pH 7.5 with 4M NaOH). Samples were vortexed andthen centrifuged in eppendorf tubes for 5 min. at 10,000 g. Thesupernatants were decanted and resuspended in 1 ml of flashtrack washsolution. The pellet was resuspended in 200 μl TB SDB and vortexed. 50μl of each sample was added to a TB lysing tube containing 0.17 g of0.2-0.3 mm glass beads, 110 μl 5% w/v sorbitol, and 300 μl of TB SDB andglass beads.

Fluid specimens such as cerebrospinal fluid (CSF) were concentrated bycentrifugation in the same manner, the supernatant decanted, and 50 μlof sediment was added to a TB lysing tube containing 0.17 g of 0.2-0.3mm glass beads, 110 μl 5% w/v sorbitol, and 300 μl of TB SDB.

For measurement from cultures, 1 μl loopful of fungal cells were addedto a TB lysing tube containing 0.17 g of 0.2-0.3 mm glass beads, 110 μl5% w/v sorbitol, and 300 μl of TB SDB.

Once samples were added to the TB lysing tube, samples from sputum andother fluids were treated in the same manner. The samples were sonicatedin a waterbath for 15 minutes at room temperature. Next the cells wereheatkilled in a heat block for 10 min. at 95° C. Dilutions were thenmade in TB SDB. Samples were used promptly or frozen at -70° C. forlater use.

Example B

Amplified Assay for Coccidioides immitis.

The assay illustrated in this section utilizes an isothermaltarget-based, transcription mediated amplification (TMA) system.Following lysis of the fungal organisms by sonication in an enclosedtube using a water bath sonicator, the released rRNA was amplified viathe TMA method described below into numerous target sequences which werethen detected in the same tube using a homogenous protection assay.

Liquid primers were diluted 30 pmols each/25 μl of a solution of PAR (4mM ATP, 4 mM CTP, 4 mM GTP, 4 mM UTP, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1mM dTTP, 40 mM Trizma Base, 20 mM MgCl₂, 17 mM KCl, 5% w/vpolyvinylpyrrolidone, adjusted to pH 7.5 using 1M NaOH and 6M HCl). 25μl of the primers in PAR were added to the bottom of a polypropylenetest tube. 200 μl of the oil reagent were added to each tube. 50 μl ofrRNA diluted to the correct concentration in TB SDB were added to eachtube (except for the negative controls which received 50 μl of SDBonly). Samples were then placed in a heat block at 95° C. for 10 min.The tubes were placed in a water bath at 42° C. for 5 min. 25 μlcontaining reverse transcriptase (458 units/μl and T7 RNA polymerase(367 units/μl) in enzyme dilution buffer (60 mM N-acetyl-L- cysteine, 1mM EDTA (free acid), 0.01% Phenol Red (0.5% solution), 140 mM TrizmaBase, 70 mM KCl, 20% v/v glycerol, and 10% v/v Triton X-102 adjusted topH 8 with 6M HCl) was added to each tube while the tubes remained in the42° C. water bath. The rack was gently shaken, the tubes were coveredwith sealing cards, and the tubes were incubated at 42° C. for anadditional 2 hours. The tubes were removed from the water bath, and thehybridization probe assay was performed. Alternatively, samples could berefrigerated for later testing.

Two primers were used in the amplification step: a T7 RNA polymerasepromoter/primer, and a primer. The T7 promoter-primer was complementaryto a region of the sense strand 3' of the hybridization probe targetsequence. Extension of this primer gives rise to RNA copies of thetarget sequence.

The primer is complementary to a region of the anti-sense strand 3' fromthe target sequence. Extension of the primer on the anti-sense strandtherefore gives rise to copies of the sense strand "template" for the T7RNA polymerase. Amplification of antisense target sequences by TMA thusoccurred as the sense strand was first copied by the DNA polymeraseactivity of reverse transcriprase and second, these sense strand copieswere used as templates by RNA polymerase to amplify the anti-sensestrand nucleic acids containing the target sequence. The process repeatsautocatalytically. Amplification of rRNA from clinical specimens is alsodescribed in Jonas et al., Detection and Identification of Mycobacteriumtuberculosis Directly from Sputum Sediments by Amplification of rRNA,pp. 2410-2416 (1993).

Example II

Hybridization Probe Assay:

A probe specific for Coccidioides immitis was identified by sequencingwith a primer complementary to the 28S rRNA of Coccidioides immitis. Theprobe SEQ ID NO:21 was characterized and shown to be specific forCoccidioides immitis. The probe distinguished Coccidioides immitis fromphylogenetically near, clinically important neighbors includingBlastomyces dermatitidis and Histoplasma capsulatum.

To demonstrate the reactivity and specificity of the probe forCoccidioides immitis, a hybridization probe assay was performed. RNA washybridized to the acridinium ester-labeled probe in the presence ofunlabeled helper probe. Helper probe consisted of the sequencecorresponding to SEQ ID N0:25: GAACAGGACG TCATAGAGGG TGAGAATCC.

Ninety μl of a probe mixture in probe hybridization buffer (100 mMsuccinic acid, 230 mM of LiOH, 150 mM aldrithiol-2, 1.2M LiCl₂, 20 mMEDTA, 20 mM EGTA, 3% v/v ethyl alcohol, 2% v/v lithium lauryl sulfateadjusted to pH 4.7 with 2M LiOH) containing 2.5 pmols helper probe and10 μl of probe (0.05 or 0.1 pmols/reaction) were added to each tube. Theprobe mixture contained an acridinium ester-labeled probes having one ofthe following nucleotide sequences:

(SEQ ID NO:17) GCAGCCACGG CATAAGTTCC TTG

(SEQ ID NO:21) GCGCCACGGC ATAAGTTCCT TG and unlabeled helper probe:

(SEQ ID N0:25) GAACAGGACG TCATAGAGGG TGAGAATCC.

The tubes were vortexed well and incubated at 60° C. for 15 min. 300 μlof 600 mM boric acid, 182 mM NaOH, and 1% v/v adjusted to pH 8.5 with 4MNaOH (selection reagent) was added to each sample. The samples were thenincubated at 60° C. for 10 min. The samples were removed from the waterbath and allowed to stand at room temperature for 5 min. before readingin a Gen-Probe luminometer for 2 seconds with a standard 2 injectionformat. A value of 30,000 relative light units (RLU) was used as theproposed cutoff for a positive test. Each run included amplificationpositive and negative controls. Quadruplicate assays were run for eachdetermination, and the mean RLU values were calculated.

Example IV

Specificity of the Amplified Hybridization Probe Assay.

Purified rRNA preparations and sonicated fungal culture lysates wereused to estimate the specificity of the test. Two probes were tested inthe amplification assay: SEQ ID NO: 17 (Tables 1A-C) and SEQ ID NO: 21(Table 1D-E). A T7 promoter-primer containing the sequence correspondingto SEQ ID NO:13 and a primer containing the sequence provided by SEQ IDNO:9 were used to obtain the results in Tables 1A and 1C. A T7promoter-primer containing the sequence corresponding to SEQ ID NO:5 anda primer sequence containing the sequence provided by SEQ ID NO:9 wereused to obtain the results in Table 1B. Strains representing othersingle pathogens and closely related organisms were included in thepanel. Only Coccidioides immitis strains were positive (>30,000 RLU) inthe assay.

The following data show that the probes did not cross react withorganisms from a wide phylogenetic cross section. The samples were alsotested with a probe which has a very broad specificity. A positivesignal from this probe provided confirmation of sample adequacy (datanot shown).

                  TABLE IA                                                        ______________________________________                                        Specificity of Hybridization Probe SEQ ID NO:17 With                          rRNA Templates                                                                ATCC             rRNA         Average                                         Organism         Concentration                                                                              RLU's                                           ______________________________________                                        (-) Control       0 fg        1,615                                           Coccidioides immitis (+) cont                                                                  500 fg       391,321                                         Malbranchea albolutea                                                                          500 fg       1,264                                           Blastomyces dermatitidis                                                                       500 fg       1,247                                           Histoplasma capsulatum                                                                         500 fg       1,171                                           Auxarthron thaxteri                                                                            500 fg       4,613                                           Gymnoascus dugwayensis                                                                         500 fg       1,255                                           (-) cont          0 fg        5,867                                           Coccidioides immitis (+) cont                                                                   50 fg       450,393                                         Coccidioides immitis (+) cont                                                                  500 fg       737,434                                         Aspergillus flavus                                                                             500 fg       2,432                                           Myxotrichum deflexum                                                                           500 fg       1,610                                           Aspergillus niger                                                                              500 fg       2,307                                           Candida krusei   500 fg       1,793                                           Candida glabrata 500 fg       5,022                                           Aspergillus fumigatus                                                                          500 fg       2,574                                           Arachnioites flavoluteus                                                                       500 fg       2,929                                           Candida parapsilosis                                                                           500 fg       2,100                                           Oidiodendron echinulatum                                                                       500 fg       1,247                                           Candida albicans 500 fg       3,189                                           ______________________________________                                         *The RLU's are averages of 4 replicates except for the value for Candida      glabrata which is the average of 2 replicates. The primers used were the      T7 promoterprimer SEQ ID NO:13 and the primer SEQ ID NO:9.               

                  TABLE IB                                                        ______________________________________                                        Specificity of Hybridization Probe (SEQ ID NO:17) With                        rRNA Templates                                                                ATCC             rRNA         Average                                         Organism         Concentration                                                                              RLU's                                           ______________________________________                                        (-) cont          0 fg        2,977                                           Coccidioides immitis (+) cont                                                                  500 fg       604,535                                         Malbranchea albolutea                                                                          500 fg       1,247                                           Blastomyces dermatitidis                                                                       500 fg       2,224                                           Histoplasma capsulatum                                                                         500 fg       968                                             Auxarthron thaxteri                                                                            500 fg       1,072                                           Gymnoascus dugwayensis                                                                         500 fg       7,638                                           (-) cont          0 fg        2,421                                           Coccidioides immitis (+) cont                                                                   50 fg       618,916                                         Coccidioides immitis (+) cont                                                                  500 fg       652,483                                         Aspergillus flavus                                                                             500 fg       2,475                                           Myxotrichum deflexus                                                                           500 fg       1,572                                           Aspergillus niger                                                                              500 fg       1,575                                           Candida krusei   500 fg       1,702                                           Candida glabrata 500 fg       2,234                                           Candida albicans 500 fg       1,607                                           Arachnioites flavoluteus                                                                       500 fg       1,521                                           Aspergillus fumigatus                                                                          500 fg       2,945                                           Candida parapsilosis                                                                           500 fg       2,210                                           Oidiodendron echinulatum                                                                       500 fg       5,726                                           ______________________________________                                         *The RLU's are the averages of 4 replicates except for Aspergillus flavus     Candida krusei and Candida galbrata which are the average of 3 replicates     The T7 promoter/primer which was used contained the sequence provided by      SEQ ID NO:5 and the primer which was used contained the sequence in SEQ I     NO:9.                                                                    

                  TABLE 1C                                                        ______________________________________                                        Specificity Testing with ATCC Cell Lysates and rRNA*                          Organism           ATCC#**   Average RLU                                      ______________________________________                                        Coccidioides immitis                                                                             46900     1,544,446                                        Coccidioides immitis                                                                             38149     1,496,889                                        Coccidioides immitis                                                                             28868     1,424,771                                        Coccidioides immitis                                                                             38146     1,478,833                                        Coccidioides immitis (1 × 10-2)                                                            38146     1,612,038                                        Coccidioides immitis (1 × 10-5)                                                            38146     1,395,387                                        Coccidioides immitis (1 × 10-9)                                                            38146     810,204                                          Histoplasma capsulatum                                                                           11407     968                                              Histoplasma capsulatum                                                                           38904     1,110                                            Blastomyces dermatitidis                                                                         60916     1,483                                            Trichophyton terrestre                                                                           28188     2,865                                            Trichophyton rubrum                                                                              10218     4,978                                            Trichophyton rubrum                                                                              28188     2,865                                            Trichophyton rubrum                                                                              CI-4373   3,369                                            Uncinocarpus reeseii                                                                             34533     1,891                                            Gymnoascus dugwayensis                                                                           18899     1,621                                            Arachnioitus flavoluteus                                                                         28364     10,336                                                              28364     1,903                                            Malbranchea dendritica                                                                           34527     4,747                                            Malbranchea arcuata                                                                              34523     1,500                                            Malbranchea albolutea                                                                            34522     1,871                                            Malbranchea gypseum                                                                              24102     4,338                                            Myxotrichum deflexum                                                                             15686     2,143                                            Auxarthron thaxteri                                                                              15598     1,414                                            Oidiodendron echinulatum                                                                         16287     1,497                                            Aspergillus flavus 10124     2,432                                            Aspergillus niger  16888     2,307                                            Aspergillus fumigatus                                                                            16907     2,574                                            Candida krusei      6258     1,793                                            Candida glabrata   48435     2,234                                            Candida albicans   18804     3,189                                            Candida parapsilosis                                                                             22019     2,100                                            Negative control             1,364                                            Coccidioides immitis (+) control                                                                 CI-W95    1,501,719                                        Trichophyton terrestre                                                                           CI-1441   1,859                                            Trichophyton terrestre                                                                           CI-TR-9   2,839                                            Trichophyton terrestre                                                                           CI-TR-73  2,924                                            ______________________________________                                         *These assays were carried out using a T7 promoterprimer containing the       sequence provided by SEQ ID NO:13, an amplification primer containing the     sequence provided by SEQ ID NO:9, and the hybridization assay probe           containing the sequence provided by SEQ ID NO:17.                             **For some organisms, the clinical isolate (CI) number is provided instea     of the ATCC number.                                                      

                  TABLE ID                                                        ______________________________________                                        Specificity of Hybridization Probe SEQ ID NO:21 With                          rRNA Templates*                                                               ATCC                rRNA        Average                                       Organism            Concentration                                                                             RLU's                                         ______________________________________                                        (-) Control         0 fg        938                                           Coccidioides immitis "A" Endospore                                                                9 × 10.sup.5                                                                        590,111                                       Coccidioides immitis "A" Endospore                                                                9 × 10.sup.3                                                                        454,899                                       Coccidioides immitis "A" Endospore                                                                9           540,528                                       Coccidioides immitis "A" Endospore                                                                0.9         348,205                                       Malbranchea dendritica                                                                            500 fg      1,264                                         Oidiodendron echinulatum                                                                          500 fg      1,247                                         ______________________________________                                         *The T7 promoter/primer used contained the sequence corresponding to SEQ      ID NO:13, and the primer used contained the sequence corresponding to SEQ     ID NO:9.                                                                 

                  TABLE 1E                                                        ______________________________________                                        Specificity of Hybridization Probe SEQ ID NO:21 With                          rRNA Templates*                                                               ATCC               rRNA        Average                                        Organism           Concentration                                                                             RLU's                                          ______________________________________                                        (-) cont            0 fg       21,616                                         Coccidioides immitis (+) control                                                                 100 fg      96.453                                         Coccidioides immitis (+) control                                                                 100 fg      143,076                                        Coccidioides immitis (+) control                                                                 200 fg      137,590                                        Coccidioides immitis (+) control                                                                 500 fg      84,636                                         Malbranchea albolutea                                                                            500 fg      13,873                                         Blastomyces dermatitidis                                                                         500 fg      11,802                                         Histoplasma capsulatum                                                                           500 fg      10,790                                         Auxarthron thaxteri                                                                              500 fg      13,724                                         Gymnoascus dugwayensis                                                                           500 fg      10,420                                         Myxotrichum deflexum                                                                             500 fg      17,156                                         Aspergillus niger  500 fg      15,943                                         Candida krusei     500 fg      16,861                                         Candida glabrata   500 fg      12,515                                         Arachnioites flavoluteus                                                                         500 fg      13,532                                         Candida parapsilosis                                                                             500 fg      14,368                                         Oidiodendron echinulatum                                                                         500 fg      12,656                                         Candida albicans   500 fg      13,946                                         ______________________________________                                         *The primers used were the T7 promoterprimer having the sequence provided     by SEQ ID NO:13 and the primer having the sequence provided by SEQ ID         NO:9.                                                                    

The above data confirm that the probes described herein are capable ofdistinguishing Coccidioides immitis from its closely relatedphylogenetic neighbors in amplification assays.

Example V

Sensitivity of the Amplified Hybridization Probe Assay To DetectCultured Coccidioides immitis.

The sensitivity of the assay was determined by testing serial dilutionsof purified Coccidioides immitis rRNA. The test detected as little as 5fg of rRNA with an average signal of 1,265,406 RLU, a value above thelinear range of the luminometer. Two assays using different probes werecarried out. The probes contained the sequences in SEQ ID NO: 17 (Table2) and SEQ ID NO: 21 (Table 3). Another approach to determine thesensitivity of the assay was to test dilutions of 3 different endosporepreparations using two different probes: SEQ ID NO: 21 (Table 4) and SEQID NO: 17 (Table 5). The number of endospores in each test wasdetermined by direct counting using a hemocytometer. The amplified assaywas able to detect as little as one endospore per test.

To determine if the assay would produce positive RLU signals in clinicalspecimens, dilutions of endospores were added to 5 culture-negativesputum sediments previously digested according to the method of Kent, etal. Kent, P. T., Kubica G. Public Health Mycobacteriology. A Guide forthe Level III Laboratory. Atlanta, Ga.; Centers for Disease Control(1985). Again, positive signals were obtained with as few as oneendospore per test. Endospores were also seeded into aliquots ofculture-negative CSF prior to centrifugation, and as few as 3 endosporesper test produced a positive result.

                  TABLE 2                                                         ______________________________________                                        Sensitivity of Hybridization Assay Probe SEQ ID NO:17                         Using rRNA Template                                                           Amplification C. immitis     Average                                          Oligonucleotides                                                                            rRNA Concentration                                                                           RLU's                                            ______________________________________                                        SEQ ID NO:13 &                                                                               5 fg          1,265,406                                        SEQ ID NO:9                                                                   SEQ ID NO:13 &                                                                              20 fg          1,878,058                                        SEQ ID NO:9                                                                   SEQ ID NO:13 &                                                                              50 fg          1,645,021                                        SEQ ID NO:9                                                                   SEQ ID NO:13 &                                                                              100 fg         1,749,740                                        SEQ ID NO:9                                                                   SEQ ID NO:13 &                                                                              (-) cont       2,582                                            SEQ ID NO:9                                                                   SEQ ID NO:5 &  5 fg          22,979                                           SEQ ID NO:9                                                                   SEQ ID NO:5 & 20 fg          343,184                                          SEQ ID NO:9                                                                   SEQ ID NO:5 & 50 fg          489,363                                          SEQ ID NO:9                                                                   SEQ ID NO:5 & 100 fg         672,477                                          SEQ ID NO:9                                                                   SEQ ID NO:5 & (-) cont       1,867                                            SEQ ID NO:9                                                                   SEQ ID NO:1 &  5 fg          11,639                                           SEQ ID NO:9                                                                   SEQ ID NO:1 & 20 fg          7,191                                            SEQ ID NO:9                                                                   SEQ ID NO:1 & 50 fg          45,217                                           SEQ ID NO:9                                                                   SEQ ID NO:1 & 100 fg         185,162                                          SEQ ID NO:9                                                                   SEQ ID NO:1 & (-) cont       2,299                                            SEQ ID NO:9                                                                   ______________________________________                                         *The RLU's are averages of 4 replicates.                                 

                  TABLE 3                                                         ______________________________________                                        Sensitivity of Hybridization Assay Probe SEQ ID NO:17                         Using rRNA Templates                                                          Amplification C. immitis     Average                                          Oligonucleotides                                                                            rRNA Concentration                                                                           RLU's                                            ______________________________________                                        SEQ ID NO:13 &                                                                               5 fg          528,577                                          SEQ ID NO:9                                                                   SEQ ID NO:13 &                                                                              20 fg          830,764                                          SEQ ID NO:9                                                                   SEQ ID NO:13 &                                                                              50 fg          743,241                                          SEQ ID NO:9                                                                   SEQ ID NO:13 &                                                                              100 fg         780,634                                          SEQ ID NO:9                                                                   SEQ ID NO:13 &                                                                              (-) cont       1,779                                            SEQ ID NO:9                                                                   SEQ ID NO:5 &  5 fg          6,517                                            SEQ ID NO:9                                                                   SEQ ID NO:5 & 20 fg          30,320                                           SEQ ID NO:9                                                                   SEQ ID NO:5 & 50 fg          159,855                                          SEQ ID NO:9                                                                   SEQ ID NO:5 & 100 fg         238,620                                          SEQ ID NO:9                                                                   SEQ ID NO:5 & (-) cont       1,842                                            SEQ ID NO:9                                                                   SEQ ID NO:1 &  5 fg          9,611                                            SEQ ID NO:9                                                                   SEQ ID NO:1 & 20 fg          116,592                                          SEQ ID NO:9                                                                   SEQ ID NO:1 & 50 fg          139,480                                          SEQ ID NO:9                                                                   SEQ ID NO:1 & 100 fg         120,685                                          SEQ ID NO:9                                                                   SEQ ID NO:1 & (-) cont       1,658                                            SEQ ID NO:9                                                                   ______________________________________                                         *The RLU's are averages of 4 replicates.                                 

The results in Tables 2 and 3 illustrate the large difference in thelevel of signal obtained in amplification assays (as measured in RLU's)using the same primer and three different T7 promoter-primers (SEQ IDNO:13, SEQ ID NO:5, and SEQ ID NO:1). The three promoter-primers weretargeted near to each other on Coccidioides immitis 28S RNA, containedthe same promoter sequence, and had similar length primer sequences. Theamplification assays using the promoter-primer containing SEQ ID NO:13gave rise to the highest signals in the different assays. The assaysusing the promoter-primer containing SEQ ID NO:5 gave rise to highersignals than the assays using the promoter-primer SEQ ID NO:1 when theprobe containing SEQ ID NO:17 was used. The amplification assays usingthe promoter-primer containing SEQ ID NO:5 gave rise to higher signalsat 50 fg and 100 fg of rRNA than the amplification assays using thepromoter-primer containing SEQ ID NO:1 when the probe containing SEQ IDNO:21 was used.

The sequence specified in SEQ ID NO:13 is 53 nucleotides long and iscomplementary to a 26 nucleotide region which begins 3 nucleotides 3' ofthe target region targeted by the sequence SEQ ID NO:21. The sequenceprovided in SEQ ID NO:5 is 49 nucleotides long and is complementary to a22 nucleotide region which begins 15 nucleotides 3' of the target regiontargeted by the sequence SEQ ID NO:21. The sequence provided in SEQ IDNO:1 is 51 nucleotides long and is complementary to a 24 nucleotideregion which begins 23 bases 3' of the target region targeted by the SEQID NO:21. Thus, the results achieved using the three promoter primersare very different, despite the similarity between the threepromoter-primers.

                  TABLE 4                                                         ______________________________________                                        Sensitivity of Hybridization Assay Probe (SEQ ID NO:21)                       Using Endospore and Spherule Dilutions*                                       Sample              Endospore Average                                         Type                Input     RLU's                                           ______________________________________                                        "A" Endospore       9 × 10.sup.5                                                                      590,111                                         "A" Endospore       9 × 10.sup.3                                                                      454,899                                         "A" Endospore       9         540,528                                         "A" Endospore       0.9       348,205                                         "B" Endospores      6.5 × 10.sup.4                                                                    580,569                                         "B" Endospores      6.5 × 10.sup.2                                                                    606,883                                         "B" Endospores      6.5       415,189                                         "B" Endospores      0.65      143,655                                         "B" Endospores      0.065     2,289                                           "C" Mixed Endos & Spherules                                                                       3.25 × 10.sup.6                                                                   573,807                                         "C" Mixed Endos & Spherules                                                                       3.25 × 10.sup.4                                                                   623,849                                         "C" Mixed Endos & Spherules                                                                       3.25 × 10.sup.2                                                                   494,017                                         "C" Mixed Endos & Spherules                                                                       3.25      131,760                                         "C" Mixed Endos & Spherules                                                                       0.325     1,679                                           (-) control         None      983                                             ______________________________________                                         *The RLU's are averages of 2 replicates. The T7 promoterprimer used           contained the sequence provided by SEQ ID NO:13 and the primer used           contained the sequence provided by SEQ ID NO:9.                          

                  TABLE 5                                                         ______________________________________                                        Sensitivity of Hybridization Assay Probe (SEQ ID NO:17)                       Using rRNA Templates                                                                             RLU's                                                      ______________________________________                                        "A" Preparation                                                               Endospores per Assay                                                          9 × 10.sup.5   1619925                                                  9 × 10.sup.3   1455427                                                  9                    1163369                                                  0.9                   360861                                                  "B" Preparation                                                               Endospores per Assay                                                          6.5 × 10.sup.4 1402927                                                  6.5 × 10.sup.2 1347311                                                  6.5                  1339671                                                  0.65                  533590                                                  0.065                  3652                                                   "C" Preparation                                                               Mixed Endospores and Spherules                                                3.25 × 10.sup.6                                                                              1359644                                                  3.25 × 10.sup.4                                                                              1366980                                                  3.25 × 10.sup.2                                                                              1218693                                                  3.25                  210010                                                  0.325                  2160                                                   Negative Control       1320                                                   ______________________________________                                         *The T7 promoterprimer used contained the sequence corresponding to SEQ I     NO:13 and the primer used contained the sequence provided by SEQ ID NO:9.

Example VI

Sensitivity of the Amplified Hybridization Probe Assay in DetectingCoccidioides immitis Directly From Clinical Specimens.

The results (average of 4 replicate RLU values) with clinical specimensare shown in Table 6. Five homogenized lung and pleural tissue specimensfrom 4 patients gave positive results from 176,235 to 1,475,187 RLU's.Four respiratory secretion specimens (sputum, bronchial washings andchest fluid) from 3 patients gave positive results from 86,998 to1,342,617 RLU's. One patient with disseminated coccidioidomycosisproduced positive signals of 569,969 and 953,985 RLU from swabs ofabscess drainage. All of these specimens were positive for C. immitis byfungal culture and/or histopathology.

Two CSF specimens and one sputum specimen from three patients werenegative by the prototype test. The RLU values ranged from 1,747 to12,850. These specimens were culture-negative for C. immitis.

Additional results confirming the detection of Coccidioides immitis inclinical specimens are shown in Tables 7-9. The probe used in theseexperiments was SEQ ID NO:17. The T7 promoter-primer used contained thesequence corresponding to SEQ ID NO: 13 and the primer used containedthe sequence corresponding to SEQ ID NO: 9. Coccidioides immitis wasdetected in pleural tissue, lung abscess tissue, pleural mass, lungmass, lung fluid, a sputum sample, and bronchial wash (Tables 6 and 7).In addition, Coccidioides immitis was detected in samples from abscesses(Table 8).

                  TABLE 6                                                         ______________________________________                                        Patient Specimens                                                             Patient #  Specimen      Average RLU                                          ______________________________________                                        C. immitis Culture or Histopathology Positive*:                               1          L. Pleural Tissue                                                                           941,285                                              1          L. Pleural Mass                                                                             1,441,898                                            2          L. Lung Abscess                                                                             176,235                                              3          L. Lung Mass  1,475,187                                            4          R. Lung Fluid 1,315,485                                            5          Sputum        1,009,956                                            6          Bronchial Wash                                                                              1,342,617                                            7          Upper Chest Fluid                                                                           258,259                                              7          Lower Chest Fluid                                                                           86,998                                               8          Lateral Leg Abscess                                                                         953,985                                              8          Medial Leg Abscess                                                                          569,969                                              C. immitis Culture Negative Specimens:                                        9          CSF           12,850                                               10         CSF           1,747                                                11         Sputum Sediment                                                                             6,786                                                ______________________________________                                         *The probe used contained the sequence in SEQ ID NO:17. The T7                promoterprimer used contained the sequence provided by SEQ ID NO:13 and       the primer used contained the sequence provided by SEQ ID NO:9.          

                  TABLE 7                                                         ______________________________________                                        PATIENT SAMPLES*                                                              Sample Type         Average rlu's                                             ______________________________________                                        L. Pleural Tissue T40995                                                                          941,285                                                   T40995 (10.sup.-1)  4,043                                                     Lung Abscess Tissue F44901                                                                        176,235                                                   F44901 (10.sup.-1)  294,295                                                   L. Pleural Mass T40996                                                                            1,441,898                                                 T40996 (10.sup.-1)  1,593,774                                                 Left Lung Mass W46404                                                                             1,475,187                                                 W46404 (10.sup.-1)  1,549,916                                                 Right Lung Fluid 94441230212                                                                      1,315,485                                                 94441230212 (10.sup.-1)                                                                           198,645                                                   Sputum Sample PN114 1,009,956                                                 PN114 (10.sup.-1)   633,191                                                   Bronchial Wash W49281                                                                             1,342,617                                                 W49281 (10.sup.-1)  889,543                                                   (-) Control         1,052                                                     (+) Control         1,763,026                                                 ______________________________________                                         *The T7 promoterprimer used in these assays contained the sequence            provided by SEQ ID NO:13, and the primer used contained the sequence          corresponding to SEQ ID NO:9. The probe used contained the sequence           provided by SEQ ID NO:17.                                                

                  TABLE 8                                                         ______________________________________                                        PATIENT SAMPLES*                                                              Sample Number      Average RLU's                                              ______________________________________                                        L. Pleural Tissue  5,766                                                      L. Pleural Tissue (10.sup.-1)                                                                    3,554                                                      L. Pleural Tissue (10.sup.-2)                                                                    2,832                                                      L. Pleural Tissue (10.sup.-3)                                                                    2,583                                                      Lung Abscess Tissue                                                                              208,062                                                    Lung Abscess Tissue (10.sup.-1)                                                                  113,559                                                    Lung Abscess Tissue (10.sup.-2)                                                                  7,663                                                      Lung Abscess Tissue (10.sup.-3)                                                                  3,406                                                      L. Pleural Mass    1,741,846                                                  L. Pleural Mass (10.sup.-1)                                                                      1,777,578                                                  L. Pleural Mass (10.sup.-2)                                                                      852,297                                                    L. Pleural Mass (10.sup.-3)                                                                      265,296                                                    Left Lung Mass     962,209                                                    Left Lung Mass (10.sup.-1)                                                                       1,260,207                                                  Left Lung Mass (10.sup.-2)                                                                       4,592                                                      Left Lung Mass (10.sup.-3)                                                                       3,920                                                      Right Lung Fluid   179,199                                                    Right Lung Fluid (10.sup.-1)                                                                     238,102                                                    Right Lung Fluid (10.sup.-2)                                                                     8,203                                                      Lung Fluid (10.sup.-3)                                                                           3,007                                                      Sputum Sample      816,631                                                    Sputum Sample (10.sup.-1)                                                                        29,590                                                     Sputum Sample (10.sup.-2)                                                                        200,518                                                    Sputum Sample (10.sup.-3)                                                                        2,267                                                      Bronchial Wash     55,001                                                     Bronchial Wash (10.sup.-1)                                                                       58,067                                                     Bronchial Wash (10.sup.-2)                                                                       18,499                                                     Bronchial Wash (10.sup.-3)                                                                       12,812                                                     (+) Cont           140,045                                                    (-) Cont           2,752                                                      ______________________________________                                         *The T7 promoterprimer used in these assays contained the sequence            provided by SEQ ID NO:13. The primer used contained the sequence              corresponding to SEQ ID NO:9. The probe used contained the sequence           corresponding to SEQ ID NO:17.                                           

                  TABLE 9                                                         ______________________________________                                        Patient Samples*                                                              Sample Type       Average RLU's                                               ______________________________________                                        Elbow Abscess (Garcia)                                                                          1,527,707                                                   Medial Abscess (Garcia)                                                                         1,020,833                                                   Elbow Abscess (Garcia)                                                                          1,178,792                                                   Abscess (Knox)    1,196,953                                                   Abscess (Knox)    1,056,303                                                   (-) Cont             5,007                                                    ______________________________________                                    

Example VII

Effects of NaOH Concentration on Sensitivity.

In order to determine the effects of NaOH concentration during treatmentof sputum specimens with N-acetyl-L-cysteine-NaOH, sputum specimens fromtwo different patients were treated at 1% and 4% NaOH during this stepof the sample preparation. The results are shown below in Table 10.

                  TABLE 10                                                        ______________________________________                                        PATIENT SAMPLES                                                               TABLE XIV                                                                     NaOH Concentration                                                                             Average RLU's                                                ______________________________________                                        Patient 1:                                                                    1% NaOH          230,162                                                      1% NaOH          203,551                                                      4% NaOH          1,005,982                                                    4% NaOH          1,080,525                                                    Patient 2:                                                                    1% NaOH          13,875                                                       1% NaOH          13,175                                                       4% NaOH          157,216                                                      4% NaOH          14,530                                                       (-) Control      5,077                                                        ______________________________________                                    

Other embodiments are within the following claims. Thus while severalembodiments have been shown and described, various modifications may bemade, without departing from the spirit and scope of the presentinvention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 41                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      AATTTAATACGACTCACTATAGGGAGAGTCCAGCAGCCACAGACGGGATT50                          C51                                                                           (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      GAATCCCGTCTGTGGCTGCTGGACTCTCCCTATAGTGAGTCGTATTAAAT50                          T51                                                                           (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      AAUUUAAUACGACUCACUAUAGGGAGAGUCCAGCAGCCACAGACGGGAUU50                          C51                                                                           (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      GAAUCCCGUCUGUGGCUGCUGGACUCUCCCUAUAGUGAGUCGUAUUAAAU50                          U51                                                                           (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      AATTTAATACGACTCACTATAGGGAGACACAGACGGGATTCTCACCCTC49                           (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      GAGGGTGAGAATCCCGTCTGTGTCTCCCTATAGTGAGTCGTATTAAATT49                           (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      AAUUUAAUACGACUCACUAUAGGGAGACACAGACGGGAUUCUCACCCUC49                           (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      GAGGGUGAGAAUCCCGUCUGUGUCUCCCUAUAGUGAGUCGUAUUAAAUU49                           (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      GCTCAAATTTGAAATCTGTCCATGCGGAGC30                                              (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     GCTCCGCATGGACAGATTTCAAATTTGAGC30                                              (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     GCUCAAAUUUGAAAUCUGUCCAUGCGGAGC30                                              (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     GCUCCGCAUGGACAGAUUUCAAAUUUGAGC30                                              (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     AATTTAATACGACTCACTATAGGGAGAGGATTCTCACCCTCTATGACGTC50                          CTG53                                                                         (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     CAGGACGTCATAGAGGGTGAGAATCCTCTCCCTATAGTGAGTCGTATTAA50                          ATT53                                                                         (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     AAUUUAAUACGACUCACUAUAGGGAGAGGAUUCUCACCCUCUAUGACGUC50                          CUG53                                                                         (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     CAGGACGUCAUAGAGGGUGAGAAUCCUCUCCCUAUAGUGAGUCGUAUUAA50                          AUU53                                                                         (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     GCAGCCACGGCATAAGTTCCTTG23                                                     (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     CAAGGAACTTATGCCGTGGCTGC23                                                     (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     GCAGCCACGGCAUAAGUUCCUUG23                                                     (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     CAAGGAACUUAUGCCGUGGCUGC23                                                     (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     GCGCCACGGCATAAGTTCCTTG22                                                      (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                     CAAGGAACTTATGCCGTGGCGC22                                                      (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                     GCGCCACGGCAUAAGUUCCUUG22                                                      (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                     CAAGGAACUUAUGCCGUGGCGC22                                                      (2) INFORMATION FOR SEQ ID NO: 25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                     GAACAGGACGTCATAGAGGGTGAGAATCC29                                               (2) INFORMATION FOR SEQ ID NO: 26:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                     GGATTCTCACCCTCTATGACGTCCTGTTC29                                               (2) INFORMATION FOR SEQ ID NO: 27:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                     GAACAGGACGUCAUAGAGGGUGAGAAUCC29                                               (2) INFORMATION FOR SEQ ID NO: 28:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                     GGAUUCUCACCCUCUAUGACGUCCUGUUC29                                               (2) INFORMATION FOR SEQ ID NO: 29:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                     GTCCAGCAGCCACAGACGGGATTC24                                                    (2) INFORMATION FOR SEQ ID NO: 30:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                     GAATCCCGTCTGTGGCTGCTGGAC24                                                    (2) INFORMATION FOR SEQ ID NO: 31:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 31:                                     GUCCAGCAGCCACAGACGGGAUUC24                                                    (2) INFORMATION FOR SEQ ID NO: 32:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 32:                                     GAAUCCCGUCUGUGGCUGCUGGAC24                                                    (2) INFORMATION FOR SEQ ID NO: 33:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 33:                                     CACAGACGGGATTCTCACCCTC22                                                      (2) INFORMATION FOR SEQ ID NO: 34:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 34:                                     GAGGGTGAGAATCCCGTCTGTG22                                                      (2) INFORMATION FOR SEQ ID NO: 35:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                     CACAGACGGGAUUCUCACCCUC22                                                      (2) INFORMATION FOR SEQ ID NO: 36:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                     GAGGGUGAGAAUCCCGUCUGUG22                                                      (2) INFORMATION FOR SEQ ID NO: 37:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 37:                                     GGATTCTCACCCTCTATGACGTCCTG26                                                  (2) INFORMATION FOR SEQ ID NO: 38:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 38:                                     CAGGACGTCATAGAGGGTGAGAATCC26                                                  (2) INFORMATION FOR SEQ ID NO: 39:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 39:                                     GGAUUCUCACCCUCUAUGACGUCCUG26                                                  (2) INFORMATION FOR SEQ ID NO: 40:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 40:                                     CAGGACGUCAUAGAGGGUGAGAAUCC26                                                  (2) INFORMATION FOR SEQ ID NO: 41:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 41:                                     AATTTAATACGACTCACTATAGGGAGA27                                                 __________________________________________________________________________

We claim:
 1. A nucleic acid hybridization assay probe for detectingCoccidioides immitis, comprising a nucleic acid 22-100 nucleotide basesin length comprising a nucleotide base sequence selected from the groupconsisting of: SEQ ID NO: 21, 22, 23, and 24;wherein said probedistinguishes Coccidioides immitis nucleic acid from Histoplasmacapsulatum and Blastomyces dermatitidis nucleic acid under stringenthybridization assay conditions.
 2. The nucleic acid hybridization assayprobe of claim 1, wherein said probe contains a detectable label.
 3. Thenucleic acid hybridization assay probe of claim 1, consisting of saidnucleotide base sequence.
 4. A nucleic acid hybrid for specificdetection of Coccidioides immitis comprising:a nucleic acidhybridization assay probe comprising a nucleic acid 22-100 nucleotidebases in length comprising a nucleotide base sequence selected from thegroup consisting of: SEQ ID NO: 21, 22, 23, and 24; and a complementarytarget sequence of Coccidioides immitis, wherein said target sequence issufficiently complementary to said probe to allow detection ofCoccidioides immitis.
 5. A probe mix comprising:a nucleic acidhybridization assay probe comprising a 22-100 base pair sequencecomprising a nucleotide sequence selected from the group consisting of:SEQ ID NOs: 21, 22, 23, and 24; wherein said probe distinguishesCoccidioides immitis nucleic acid from Histoplasma capsulatum andBlastomyces dermatitidis nucleic acid under stringent hybridizationassay conditions, and a helper probe comprising a nucleic acid sequenceselected from the group consisting of: SEQ ID NOs: 25, and
 27. 6. Amethod for detecting the presence of Coccidioides immitis in a testsample and distinguishing said Coccidioides immitis from Histoplasmacapsulatum and Blastomyces dermatitidis, comprising the steps of:a)contacting said test sample under stringent hybridization assayconditions with a nucleic acid hybridization assay probe which forms ahybrid stable for detection under stringent hybridization conditionswith Coccidioides immitis nucleic acid, and said probe comprising anucleic acid 22-100 nucleotide bases in length comprising a nucleotidebase sequence selected from the group consisting of: SEQ ID NOs: 21, 22,23, and 24; wherein said probe distinguishes Coccidioides immitisnucleic acid from Histoplasma capsulatum and Blastomyces dermatitidisnucleic acid under stringent hybridization assay conditions; and (b)detecting said hybrid as an indication of the presence of Coccidioidesimmitis in said sample.
 7. The method of claim 6, further comprising theuse of at least one helper oligonucleotide selected from the groupconsisting of: SEQ ID NOs: 25 and
 27. 8. The method of claim 6, furthercomprising the step of amplifying said Coccidioides immitis targetnucleic acid prior to said step (a).
 9. The method of claim 8, whereinsaid amplifying uses an amplification oligonucleotide from 15 to 100nucleotides in length able, under nucleic acid amplification conditions,to bind to or extend through a nucleotide base sequence selected fromthe group consisting of SEO ID NOs. 10, 12, 30, 32, 34, 36, 38 and 40.10. The method of claim 9, wherein said oligonucleotide comprises asequence selected from the group consisting of: SEQ ID NOs: 5, 33, and35.
 11. The method of claim 9, wherein said oligonucleotide comprises asequence selected from the group consisting of: SEQ ID NOs: 13, 37, and39.
 12. The method of claim 9, wherein said sample is a clinical sampleand said method is used to detect and distinguish Coccidioides immitisfrom Histoplasma capsulatum, Blastomyces dermatitidis, Oidiodendronechinulatum, and Malbranchea dendriticus, if present, directly from aclinical specimen.
 13. The method of claim 8, wherein said amplifyinguses an amplification oligonucleotide consisting of a 22-100 basesequence having a nucleotide sequence selected from the group consistingof: SEQ ID NOs: 29, 31, 33, 35, 37, and
 39. 14. The method of claim 13,wherein said amplification oligonucleotide further comprises at its 5'end a nucleic acid sequence recognized by an RNA polymerase or whichenhances initiation or elongation by an RNA polymerase.
 15. The methodof claim 9, wherein said amplification oligonucleotide is recognized bysaid RNA polymerase.
 16. A method for detecting the presence ofCoccidioides immitis in a test sample and distinguishing saidCoccidioides immitis from Histoplasma capsulatum and Blastomycesdermatitidis, comprising the steps of:(a) amplifying Coccidioidesimmitis target nucleic acid in a test sample using an amplificationoligonucleotide consisting of a 22-100 base sequence having a nucleotidesequence selected from the group consisting of: SEQ ID NOs: 29, 31, 33,35, 37, and 39; (b) contacting the amplified target nucleic acid understringent hybridization assay conditions with a nucleic acidhybridization assay probe which forms a hybrid stable for detectionunder stringent hybridization conditions with Coccidioides immitisnucleic acid, and said probe consisting of a 22-100 base sequence havinga nucleotide sequence selected from the group consisting of: SEQ IDNOs:21, 22, 23, and 24; wherein said probe can distinguish Coccidioidesimmitis nucleic acid from Histoplasma capsulatum and Blastomycesdermatitidis nucleic acid under stringent hybridization assayconditions; and (c) measuring a presence or amount of said hybrid stablefor detection.
 17. The method of claim 9, wherein said amplificationoligonucleotide comprises a sequence selected from the group consistingof: SEQ ID NOs: 1, 5, 13, 29, 31, 33, 35, 37, and
 39. 18. The nucleicacid hybridization assay probe of claim 2, wherein said detectable labelis an acridinium ester.
 19. A probe mix comprising;a) a nucleic acidhybridization assay probe for detection of Coccidioides immitis,comprising a nucleic acid 22-100 nucleotide bases in length comprising anucleotide sequence selected from the group consisting of: SEQ ID NOs:21, 22, 23, and 24; wherein said probe distinguishes Coccidioidesimmitis nucleic acid from Histoplasma capsulatum and Blastomycesdermatitidis nucleic acid under stringent hybridization assayconditions, and b) a helper probe.
 20. An amplification oligonucleotidefrom 15 to 100 nucleotides in length able, under nucleic acidamplification conditions, to bind to or extend through a nucleotide basesequence selected from the group consisting of SEQ ID NOs. 10, 12, 30,32, 34, and
 36. 21. The amplification oligonucleotide of claim 20 from15 to 50 nucleotides in length.
 22. The amplification oligonucleotide ofclaim 20, comprising a contiguous nucleotide sequence selected from thegroup consisting of SEQ ID NOs. 9, 11, 29, 31, 33, and
 35. 23. Theamplification oligonucleotide of claim 20, consisting of a contiguousnucleotide sequence selected from the group consisting of SEQ ID NOs. 9,11, 29, 31, 33, and
 35. 24. The amplification oligonucleotide of claim20 which comprises, in the 5' upstream region, an oligonucleotidesequence which is recognizable by an RNA polymerase and enhancesinitiation or elongation by said RNA polymerase.
 25. The amplificationoligonucleotide of claim 24, wherein said oligonucleotide sequencerecognizable by an RNA polymerase comprises SEQ ID NO.
 41. 26. Theamplification oligonucleotide of claim 25, comprising a sequenceselected from the group consisting of SEQ ID NOs. 1, 5 and
 13. 27. Theamplification oligonucleotide of claim 25, consisting of a sequenceselected from the group consisting of SEQ ID NOs. 1, 5 and
 13. 28. Acomposition for amplifying or detecting Coccidioides immitis nucleicacid, comprising at least one oligonucleotide selected from the groupconsisting of:(a) a first oligonucleotide from 15 to 100 nucleotides inlength which will hybridize to at least a portion of a first region ofCoccidioides immitis nucleic acid, wherein said first region consists ofa contiguous nucleotide sequence selected from the group consisting ofSEQ ID NOs. 30, 32, 34, 36, 38 and 40; and (b) a second oligonucleotidefrom 15 to 100 nucleotides in length which will hybridize to at least aportion of a second region of Coccidioides immitis nucleic acid, whereinsaid second region consists of a contiguous nucleotide sequence selectedfrom the group consisting of SEQ ID NOs. 10 and
 12. 29. The compositionof claim 28, wherein said first oligonucleotide comprises a contiguousnucleotide sequence selected from the group consisting of SEQ ID NOs.29, 31, 33, 35, 37, and 39 and said second oligonucleotide comprises acontiguous nucleotide sequence selected from the group consisting of SEQID NOs. 9 and
 11. 30. The composition of claim 28, wherein at least oneof said first oligonucleotide or said second oligonucleotide furthercomprises, in the 5' upstream region, an oligonucleotide sequence whichis recognizable by an RNA polymerase and enhances initiation orelongation by said RNA polymerase.
 31. The composition of claim 30,wherein said oligonucleotide sequence recognizable by an RNA polymerasecomprises SEQ ID NO.
 41. 32. The composition of claim 31, wherein saidfirst oligonucleotide comprises a contiguous nucleotide sequenceselected from the group consisting of SEQ ID NOs. 1, 5 and
 13. 33. Thecomposition of claim 28, further comprising a nucleic acid hybridizationprobe which comprises a third oligonucleotide from 15 to 100 nucleotidesin length which will hybridize with a region of Coccidioides immitisnucleic acid to form a detectable hybridization duplex under selectivehybridization conditions, wherein said region consists of a contiguousnucleotide sequence selected from the group consisting of SEQ ID NOs. 17and 21, and their fully complementary sequences of the same length. 34.The composition of claim 33, wherein said third oligonucleotidecomprises a contiguous nucleotide sequence selected from the groupconsisting of SEQ ID NOs. 17 and 21, and their fully complementarysequences of the same length.
 35. The composition of claim 33, whereinsaid third oligonucleotide consists of a contiguous nucleotide sequenceselected from the group consisting of SEQ ID NOs. 17 and 21, and theirfully complementary sequences of the same length.
 36. The composition ofclaim 33, wherein said first oligonucleotide consists of SEQ ID NO. 1,5, or 13, said second oligonucleotide consists of SEQ ID NO. 9, and saidthird oligonucleotide consists of SEQ ID NO. 17 or
 21. 37. Thecomposition of claim 33, wherein said probe contains a detectable label.38. The composition of claim 37, wherein said detectable label is anacridinium ester.
 39. The composition of claim 28, further comprising atleast one helper oligonucleotide.
 40. The composition of claim 38,wherein said helper oligonucleotide consists of SEQ ID NO. 25 or 27.